As our knowledge of the genome expands, so does our understanding of the characteristics of what we define as genes. Small Open Reading Frame (smORF) genes have eluded gene annotation until very recently, and evidence is mounting that these very short nucleotide sequences encode functional peptides that are ≤100 amino acids in size. From work conducted in the fruit fly, our lab has successfully characterised three Drosophila smORFs, of which two have been shown to have a function in higher vertebrates, including humans. The functional characterisation of one of these conserved smORF encoded peptides (SEPs), Hemotin, is presented in this thesis. Though the overall number is still low compared to the abundance of potential smORF-encoding genes in Drosophila, the information gathered here allows us to speculate on the wider role of smORF peptides through cell-based imaging studies conducted on Drosophila cells. Here, I will discuss the various techniques that can and should be employed in order to study the functions of SEPs. Chapter III describes the various phenotypic studies conducted on the Hemotin smORF which is expressed in Drosophila haemocytes, and are integral to the fruit fly immune system. This study showed that connecting subcellular localisation of an SEP to a direct functional assay in cells can reveal functional characteristics of the peptide for further study. Chapter IV details the results from a tagging-transfection assay, which began initially as a way to independently corroborate the translation of smORF mRNAs that were assessed as such by Ribosome Profiling. This experiment resulted in the discovery of several mitochondrial-localised SEPs in Drosophila S2 cells, opening the door for the direct functional assay described in Chapter V. The results from a small-scale RNAi screen conducted on the mitochondria of S2 cells provided a reliable read-out for functionality of a large proportion of the smORFs that were screened. This assay can potentially be used as a phenotypic read-out of mitochondrial-SEP function in any cell or tissue type. Elucidation of smORFs and the functions of the peptides that they encode will help us to expand the Drosophila proteome, along with providing evidence of their functionality across every organism in which they are found. Considering that characterised SEPs play very important roles in physiology and health, it is time for smORFs to be acknowledged as the important genomic elements that they are.